The goal of this project is to use in vitro studies in cell lines and primary human cells and human cervicovaginal tissue explants to test candidate siRNA complexes for their ability to target different cell types potentially important in HIV transmission (Langerhans cells and lamina propria T cells, dendritic cells and macrophages), efficiently induce gene silencing and provide protection from challenge with HIV, all without unintended toxicity. siRNAs targeting viral coreceptors and/or HIV genes can prevent viral entry or suppress HIV replication, even in productively infected cells. siRNAs targeting more than one gene have a synergistic effect on blocking HIV replication. A key lesson learned from efforts to develop effective HIV drug regimens is that to control the virus it is necessary to target multiple steps in its lifecycle. One of the advantages of using siRNAs is that it is relatively easy to develop a cocktail of siRNAs that can simultaneously target several genes. HIV can mutate to escape from inhibition by siRNAs, just as it can escape from conventional drugs. One aim of this project is to identify siRNAs that might be included in a cocktail to silence other host genes besides CCR5, as well as conserved HIV target sequences that will provide cross-clade protection and be difficult for the virus to mutate without a loss in fitness. In addition siRNA sequences against macaque CCR5 and the SHIV challenge isolate will be identified for rhesus challenge studies in Project by Veazey. This project will use engineered vaginal tissue and cervicovaginal explants to verify that Langerhans cells and other cells important in HIV transmission are effectively transduced and silenced by siRNA-lipid complexes and protected from HIV transmission. If not, it will work closely with the siRNA Manufacturing and Toxicology Core to design and test modified siRNAs or delivery complexes for enhanced transduction, silencing and protection, using strategies developed by the investigators that have been validated in vivo. As siRNAs are optimized as to sequence, chemical modification for stability and delivery, and formulation, they will initially be tested in primary human cells and tissue models in this project, before testing in mice or macaques in Projects by Palliser and Veazey.